The present invention relates to hybrid bacterial trains which may be used for the expression of high levels of Vibrio cholerae O-somatic antigen (Vc OAg).
The genes encoding the O-antigen from both serotypes of Vibrio cholerae have been cloned into E.coli K-12. Such plasmid clones are pPM1001, pPM1002, pPM1003, and pPM1004 which is derived from pPM1001. [These plasmids have been described in a previous patent application PCT/AU85/00015, now allowed U.S. Ser. No. 07/080,362, the entire disclosure of which is incorporated herein by reference, and in Manning et al, (1986) Infect. Immun 53(2) 272-277]. E.coli K-12 strains harbouring these plasmids produce high levels of Vc OAg which is in the form of a lipopolysaccharide (LPS) since it is extractable by the standard hot phenol/water method, is detectable by silver staining of cell envelope material electrophoresed on a SDS-polyactylamide gel, and it is on the cell surface of clones as judged by agglutination with antiserum to Vibrio cholerae.
However, when pPM1004 is introduced into either Salmonella typhimurium strain G30, or into Salmonella typhi strain Ty21a, both galE mutant strains, the Vc OAg is produced at low levels and is not in the form of a lipopolysaccharide since it cannot be extracted by the hot phenol/water method and cannot be silver stained as described above. Since these, and other Salmonella strains, express the Vc OAg poorly, then this is a major obstacle to the use of Salmonellae as a carrier strain for Vc OAg in a live oral vaccine; Vc OAg is known to be a protective antigen for immunity to cholera.
Poor expression of Vc OAg in Salmonella relative to the expression obtained in E.coli K-12 may be the result of any one of the many differences between the two species. The block (or blocks) in expression may occur at the level of gene expression or biosynthesis and assembly.
It is accordingly an object of the present invention to overcome, or at least alleviate one or more of the difficulties related to the prior art.